Important advances have been made in the treatment of viral infections over the last two decades, especially with the extensive availability of drugs to treat HIV infection, but there is a need to develop new drugs particularly with the challenges facing HIV vaccine development and the propensity to develop drug resistance. With FDA approval in 2007 of the first antiretroviral drug targeting a cellular gene, the HIV coreceptor CCR5, there is now proof of concept that a cellular target can be used to develop a highly effective, safe, nontoxic and well-tolerated antiretroviral drug. Although somewhat of a paradigm shift for infectious diseases, in fact, cellular genes are targeted routinely for non-infectious diseases. In any case this demonstrates the potential for targeting cellular genes for antiviral therapy. This proposal aims to extend the work accomplished over the last several years using a proprietary technology, gene-trap, which is a rapid and efficient method for the identification of host genes that are necessary for infection, replication or pathogenesis by viruses. Gene-trap insertional mutagenesis has identified cells with genes that are not expressed;loss of these genes allows the cell to survive during lethal virus infection, but apparently is not required for cell survival. To date, we have identified several pathways used in common by a number of viruses, suggesting that there may be key cellular mechanisms necessary for pathogenesis and that blocking such mechanisms in vivo could confer broad-spectrum therapeutic intervention. Our hypothesis is that by screening siRNAs to genes identified by gene trap and confirmed by influenza virus infection and testing them in indicator cell lines and primary marrophages for HIV infection, we will discover new host gene targets that can be used to develop antiretroviral drugs. During Phase I, we will test the effects of down regulation of specific genes indicator cell lines for effect on HIV based on well-defined criteria to select lead targets for drug discovery criteria. The most important criteria is a high resolution 3D crystal structure available for the target. The importance of crystal structure is to facilitate drug discovery efforts that will begin in phase I and that will be the primary focus in phase II of the project. Using the indicator cell line U373 MAGI, which are readily transfectable with siRNA, we will test the effect on HIV replication both by beta-GAL activity, which will identify inhibition of replication at steps prior to gene expression, including entry reverse transcription and integration, and we will also measure extracellular virus by HIV p24 production and Real-time PCR for HIV RNA, to capture inhibition events that occur in the second half of the virus life cycle. By performing these two assays in parallel we determine whether the gene is important in early viral life cycle events or later ones. To confirm the relevance of these genes for HIV replication we will assess effects of gene down regulation on primary human macrophages, which are natural targets for infection. In independent studies to perform gene-trap and develop drugs for influenza virus infection, the genes that will be assessed for effects on HIV replication will have been assessed in parallel in mouse studies. Therefore, we will have animal safety data for these genes, with down regulation maintained in the animals for a period of one month. Genes identified as important for HIV infection, and meeting our criteria for drug development, will be screened with small molecule libraries with collaborators at Emory, lead by Dennis Liotta, PhD. This proposal will be likely to lead to a large number of new antiretroviral drugs that affect novel targets for therapy. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page PUBLIC HEALTH RELEVANCE: Tremendous advances have been made in treatment of HIV infection over the last ten years, with introduction of three new drugs in the last one to two years that are safe, well-tolerated and typically effective against virus strains resistant to older drugs. The HIV pipeline is not robust, however, and after several "me too" drugs directed to the same targets over the next several years, integrase and CCR5, there are not many other antiretroviral therapies that will be developed clinically. Thus, there is an urgent need to develop new drugs against novel targets for HIV since resistance to older drugs emerges in many patients. Our proposal will identify human proteins as targets important for HIV infection that can be used to develop new classes of antiretroviral drugs. PHS 398/2590 (Rev. 11/07) Page 1 Continuation Format Page